For patients diagnosed with solid tumors of the breast or pancreas, there's a strong correlation between disease progression events, like metastasis or therapy resistance, and poor outcomes. The cellular molecular mechanisms that govern tissue homeostasis and fibrosis have been known to also control solid tumor progression. My lab's interested in understanding these mechanisms so that we can better develop therapies and diagnostic measures to help patients.
One of the main experimental challenges is that there's a critical need for 3D cancer models that can capture intercellular interactions during physiological and pathophysiological processes, and then by understanding these interactions, additional insights into the diseases can be understood to then develop new treatment strategies. Our protocol will provide a cost-effective and reproducible 3D cell culture method that replicates in vivo tissue microenvironment features. Our protocol will provide an easy and rapid scaffold-free and scaffold-based 3D cell culture methods in which we can quantify heterogeneous cellular interactions.
We have discovered a way to efficiently formulate 3D spheroid cultures that can be used with scaffold-free models, but also can be merged with scaffold-based systems to measure cell invasion and behavior. To begin, turn on the ultraviolet light to sanitize the interior of the biosafety cabinet for 15 minutes. Open the biosafety cabinet window sash to stabilize the airflow and turn on the vacuum aspiration system.
Clean the interior hood surface and the tubing of the vacuum aspiration system with 70%ethanol. Warm the cell culture medium, PBS, and 0.25%trypsin EDTA to 37 degrees Celsius in a bead bath. Now, examine the cells under a microscope to confirm 70 to 80%confluency.
Using a vacuum aspirator, aspirate and discard the culture medium from the plated cells. Wash the remaining medium once with two milliliters of PBS, then aspirate and discard the PBS after the wash. Next, using a micropipette, add one milliliter of trypsin to the cell culture dish and place the plate inside a 5%carbon dioxide incubator at 37 degrees Celsius for five minutes.
Now, add one milliliter of soybean trypsin inhibitor in PBS to the plate to inactivate the trypsin. To disperse the cell clusters, pipette the liquid mixture using a P-1000 micropipette. Collect the cell suspension from the bottom of the plate and transfer it to a 15 milliliter conical tube.
Centrifuge the tube at 100 G for five minutes at room temperature and discard the supernatant using the vacuum aspirator. Let the molecular fluorescent probes warm to room temperature for 15 minutes in a bead bath tempered at 37 degrees Celsius. Using a micropipette, resuspend the cells in two milliliters of the respective working cell tracker dye media solutions.
Incubate the tubes at 37 degrees Celsius in a 5%carbon dioxide incubator. After 30 minutes of incubation, centrifuge the tubes at 100 G for five minutes at room temperature. Then, use a vacuum aspirator to aspirate and discard the supernatant.
Resuspend the pellet thoroughly in one milliliter of 10%FBS-containing DMEM using a micropipette. Now, collect 10 microliters of the cell suspension and transfer it to a microtube containing an equal volume of trypan blue. After mixing the cells, add 20 microliters of the cell trypan solution to a cell counting chamber slide.
Insert the slide into an automated cell counter to determine the cell number. Calculate the average total live cell number from two readings. Prepare working cell stocks for each cell type at a concentration of 6.67 times 10 to the power of three cells per milliliter, equivalent to 2, 000 cells per 300 microliters.
Using a micropipette, transfer the required volume for three technical replicates plus one extra into a microtube. After mixing thoroughly with a pipette, transfer 300 microliters of the sample to a well of a U-shaped bottom ultra low attachment 96-well microplate. Place the 96-well plate in an incubator at 37 degrees Celsius.
Image spheroid growth and morphology every 24 hours, up to 96 hours, using a phase contrast microscope. Turn on the imaging and automated incubator devices. Create a new imaging protocol in the imaging software's task manager to capture the 48-hour growth of BT-474 spheroids co-cultured with BJ-5ta fibroblasts and EA.hy926 endothelial cells stained with the cell tracker dyes blue, orange, and deep red respectively.
Fill an ice bucket with ice to keep the basement membrane extract solution cold and place the solution on ice. Using a multichannel pipette, aspirate approximately 170 microliters of medium from the culture dish. Obtain a magnifying glass and a mini light box to closely observe the small spheroids.
Place the 96-well spheroid plate over the light box and position the magnifying glass overhead. Set a P-200 pipette to 30 microliters and collect the basement membrane extract to create three microdroplets. Ensure the 96-well plate is flat and position the pipette vertically above the spheroid.
Now, release the droplet without touching the bottom of the well. Place the plate in an incubator at 37 degrees Celsius for 20 minutes. After incubation, overlay the spheroids with an additional 50 microliters of the basement membrane extract solution per well and incubate the plate at 37 degrees Celsius for 30 minutes.
Then, using a micropipette, add 100 microliters of cell culture medium to each well. At 72 hours post-plating, MCF-10A, MCF-10Ca1H, and BT-474 cells co-cultured with EA.hy926 and THP-1 or with BJ5-ta and THP-1 exhibited a significant increase in spheroid area compared to monoculture epithelial spheroids. In contrast, co-cultured MDA-MB-468 cells showed a significant decrease in spheroid area compared to monoculture.
The application of cell tracker dye to BT-474 tumorigenic epithelial cells and stromal cells prior to spheroid establishment demonstrated that stromal cells, including EA.hy926 and BJ-5ta, formed the budding structures at the perimeter of the central BT-474 spheroids. At 24 hours post-plating, a basement membrane was overlaid on BT-474 tumorigenic epithelial cells co-cultured with stromal cells, demonstrating the formation of invasive structures.
